This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We first determined the optimal dose of the third-generation live attenuated Listeria monocytogenes vector encoding SIV gag, termed Lmdd-BdopSIVgag, in rhesus monkeys. Next, we examined different dose-schedules for intragastric vaccination. Controls received "empty" vector, Lmdd-Bdop. Every-other-day immunization was not as good as vaccinating daily for three consecutive days. To test whether prior vaccination with Listeria would tolerize, we re-immunized monkeys that had undergone a full vaccination series with the same Lmdd-BdopSIVgag with a daily x 3 schedule;this group of monkeys was designated Group 1B. A new group of 5 na[unreadable]ve monkeys was vaccinated in parallel (Group 1A). New na[unreadable]ve controls (Group 2;5 monkeys) received only empty vector. Good cellular immunity was seen in Groups 1A and 1B, effectively ruling out tolerization. Both Groups then received two mucosal boosts with live attuenated adenovirus encoding SIV Gag (Ad5hrSIVGag), which resulted in strong increases in Gag-specific cellular immunity. Next, Groups 1A and 1B received protein immunizations (trimeric HIV-1 gp160 and Tat) to induce neutralizing antibodies. At the conclusion of the protein boosts, the monkeys were challenged with 5 low, weekly intrarectal doses of an R5 SHIV encoding a heterologous HIV clade C envelope. Substantial protection was seen, including prevention of infection. We conclude that inducing balanced immune responses consisting of cellular as well as humoral immunity can be successful. Strong cellular immunity was induced with oral Lmdd-BdopSIVgag priming, followed by mucosal boosting with Ad5hrSIVGag, whereas cross-neutralzing antibodies were generated with HIV-1 gp160 and Tat.